Crystalline tripeptide epoxy ketone protease inhibitors

ABSTRACT

The invention relates to crystalline tripeptide keto epoxide compounds, methods of their preparation, and related pharmaceutical compositions.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/257,887, filed Dec. 5, 2011, which is the U.S. national stage under35 U.S.C. §371 of International Application Number PCT/US2010/028126,filed on 22 Mar. 2010, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/162,196, filed Mar. 20, 2009, and U.S.Provisional Application Ser. No. 61/180,561, filed May 22, 2009; each ofthese prior applications is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

In eukaryotes, protein degradation is predominately mediated through theubiquitin pathway in which proteins targeted for destruction are ligatedto the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinatedproteins then serve as substrates for the 26S proteasome, amulticatalytic protease, which cleaves proteins into short peptidesthrough the action of its three major proteolytic activities. Whilehaving a general function in intracellular protein turnover,proteasome-mediated degradation also plays a key role in many processessuch as major histocompatibility complex (MHC) class I antigenpresentation, apoptosis, cell growth regulation, NF-κB activation,antigen processing, and transduction of pro-inflammatory signals.

The 20S proteasome is a 700 kDa cylindrical-shaped multicatalyticprotease complex comprised of 28 subunits organized into four rings. Inyeast and other eukaryotes, 7 different α subunits form the outer ringsand 7 different β subunits comprise the inner rings. The α subunitsserve as binding sites for the 19S (PA700) and 11S (PA28) regulatorycomplexes, as well as a physical barrier for the inner proteolyticchamber formed by the two β subunit rings. Thus, in vivo, the proteasomeis believed to exist as a 26S particle (“the 26S proteasome”). In vivoexperiments have shown that inhibition of the 20S form of the proteasomecan be readily correlated to inhibition of 26S proteasome. Cleavage ofamino-terminal prosequences of β subunits during particle formationexpose amino-terminal threonine residues, which serve as the catalyticnucleophiles. The subunits responsible for catalytic activity inproteasomes thus possess an amino terminal nucleophilic residue, andthese subunits belong to the family of N-terminal nucleophile (Ntn)hydrolases (where the nucleophilic N-terminal residue is, for example,Cys, Ser, Thr, and other nucleophilic moieties). This family includes,for example, penicillin G acylase (PGA), penicillin V acylase (PVA),glutamine PRPP amidotransferase (GAT), and bacterialglycosylasparaginase. In addition to the ubiquitously expressed βsubunits, higher vertebrates also possess three interferon-γ-inducible βsubunits (LMP7, LMP2 and MECL1), which replace their normalcounterparts, β₅, β₁ and β₇ respectively, thus altering the catalyticactivities of the proteasome. Through the use of different peptidesubstrates, three major proteolytic activities have been defined for theeukaryote 20S proteasome: chymotrypsin-like activity (CT-L), whichcleaves after large hydrophobic residues; trypsin-like activity (T-L),which cleaves after basic residues; and peptidylglutamyl peptidehydrolyzing activity (PGPH), which cleaves after acidic residues. Twoadditional less characterized activities have also been ascribed to theproteasome: BrAAP activity, which cleaves after branched-chain aminoacids; and SNAAP activity, which cleaves after small neutral aminoacids. The major proteasome proteolytic activities appear to becontributed by different catalytic sites, since inhibitors, pointmutations in β subunits and the exchange of γ interferon-inducing βsubunits alter these activities to various degrees.

Improved compositions and methods for preparing and formulatingproteasome inhibitor(s) are needed.

SUMMARY OF THE INVENTION

One aspect of the invention relates to crystalline compounds having astructure of Formula (I) or a pharmaceutically acceptable salt thereof,

wherein

X is O, NH, or N-alkyl, preferably O;

Y is N, S, or C(R⁸)₂, preferably NH;

Z is NH, N-alkyl, O, S or C(R⁸)₂, preferably S;

R¹, R², and R³ are hydrogen;

-   each of R⁴, R⁵, R⁶, R⁷ and R⁸ is independently selected from    hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and    C₁₋₆aralkyl, each of which is optionally substituted with a group    selected from alkyl, amide, amine, carboxylic acid or a    pharmaceutically acceptable salt thereof, carboxyl ester, thiol, and    thioether, preferably R⁴, R⁵ and R⁶, are independently selected from    C₁₋₆thioether, C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl and R⁷ is    C₁₋₆alkyl, more preferably, R⁴ and R⁵ are C₁₋₆thioether, and R⁷ is    C₁₋₆alkyl.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a DSC (differential scanning calorimetry) thermogram ofcrystalline compound 1.

FIG. 2 shows an XRPD (X-ray powder diffraction) pattern of crystallinecompound 1.

FIG. 3 shows a TG thermogram of crystalline compound 1.

FIG. 4 shows modulated thermograms of amorphous compound 1, reversingheat flow (bottom) and non-reversing heat flow (top).

FIG. 5 shows a comparison of DSC thermograms of crystalline compound 1prepared according to Example 2 (middle), Example 3 (top), and Example 4(bottom).

FIG. 6 shows an XRPD pattern of amorphous compound 1 prepared accordingto Example 1 (bottom), as compared to XRPD patterns of crystallinecompound 1 prepared according to Example 2 (top), Example 3 (2^(nd) frombottom), and Example 4 (2^(nd) from top).

FIG. 7 shows a TG thermogram of amorphous compound 1.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, the invention relates to crystalline compoundshaving a structure of Formula (I) or a pharmaceutically acceptable saltthereof,

wherein

X is O, NH, or N-alkyl, preferably O;

Y is NH, N-alkyl, O, S, or C(R⁸)₂, preferably NH;

Z is NH, N-alkyl, O, S or C(R⁸)₂, preferably S;

R¹, R², and R³ are hydrogen;

-   each of R⁴, R⁵, R⁶, R⁷ and R⁸ is independently selected from    hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and    C₁₋₆aralkyl, each of which is optionally substituted with a group    selected from alkyl, amide, amine, carboxylic acid or a    pharmaceutically acceptable salt thereof, carboxyl ester, thiol, and    thioether, preferably R⁴, R⁵ and R⁶, are independently selected from    C₁₋₆thioether, C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl and R⁷ is    C₁₋₆alkyl, more preferably, R⁴ and R⁵ are C₁₋₆thioether, and each R⁷    is C₁₋₆alkyl

In certain embodiments, the invention relates to a crystalline compoundof Formula (II)

In certain embodiments, the invention relates to a method for thepreparation of a crystalline compound of Formula (I) or (II), comprisingone or more of: (i) preparing the amorphous compound, e.g., according toU.S. patent application Ser. No. 11/595,804; (ii) dissolving theamorphous compound in an organic solvent; (iii) bringing the solution tosupersaturation; (iv) isolating the crystals, e.g., by filtering thecrystals, by decanting fluid from the crystals, or by any other suitableseparation technique; and (v) washing the crystals. In certainembodiments, preparation further comprises inducing crystallization. Incertain embodiments, preparation further comprises drying, preferablyunder reduced pressure, such as under vacuum pressure.

In certain embodiments, the amorphous compound may be dissolved in asolvent selected from acetonitrile, ethyl acetate, heptanes, hexanes,isopropyl acetate, methanol, methylethyl ketone, tetrahydrofuran,toluene, and water, or any combination thereof. In certain embodiments,the amorphous compound of Formula (II) may be dissolved in an organicsolvent selected from acetonitrile, heptanes, hexanes, methanol,tetrahydrofuran, and toluene, or any combination thereof. In certainpreferred embodiments, the organic solvent is toluene, tetrahydrofuran,or acetonitrile, preferably acetonitrile or toluene.

In certain embodiments, bringing the solution to supersaturationcomprises the slow addition of an anti-solvent, such as water, heptanes,hexanes or another polar or non-polar liquid miscible with the organicsolvent, allowing the solution to cool (with or without seeding thesolution), reducing the volume of the solution, or any combinationthereof. In certain embodiments, bringing the solution tosupersaturation comprises adding an anti-solvent, cooling the solutionto ambient temperature or lower, and reducing the volume of thesolution, e.g., by evaporating solvent from the solution. In certainembodiments, allowing the solution to cool may be passive (e.g.,allowing the solution to stand at ambient temperature) or active (e.g.,cooling the solution in an ice bath or freezer).

In certain embodiments, the method further comprises inducingprecipitation or crystallization. In certain embodiments, inducingprecipitation or crystallization comprises secondary nucleation, whereinnucleation occurs in the presence of seed crystals or interactions withthe environment (crystallizer walls, stirring impellers, sonication,etc.).

In certain embodiments, washing the crystals comprises washing with aliquid selected from anti-solvent, acetonitrile, heptanes, hexanes,methanol, tetrahydrofuran, toluene, water, or a combination thereof. Incertain embodiments, the crystals are washed with a combination ofanti-solvent and the organic solvent. In certain embodiments, theanti-solvent is water, while in other embodiments it is an alkanesolvent, such as hexane or pentane, or an aromatic hydrocarbon solvent,such as benzene, toluene, or xylene.

In certain embodiments, washing the crystals comprises washing thecrystalline compound of Formula (II) with a mix of tetrahydrofuran andan alkane solvent, such as hexanes or heptanes, or with a mix ofacetonitrile and water. In certain embodiments, washing the crystalscomprises washing the crystalline compound of Formula (II) with toluene.In preferred such embodiments, the toluene is cooled prior to washing.

In certain embodiments, a crystalline compound of Formula (II) issubstantially pure. In certain embodiments, the melting point of thecrystalline compound of Formula (II) is in the range of about 135 toabout 160° C., about 140 to about 155° C., about 145 to about 150° C.,or even about 147 to about 149° C., e.g., about 149° C.

In certain embodiments, the DSC of a crystalline compound of Formula(II) has a sharp endothermic maximum at about 147° C., e.g., resultingfrom melting and decomposition of the crystalline form as shown in FIG.1.

In certain embodiments, the X-ray powder pattern of a crystallinecompound of Formula (II) is)(θ-2θ°): 8.94; 9.39; 9.76; 10.60; 11.09;12.74; 15.27; 17.74; 18.96; 20.58; 20.88; 21.58; 21.78; 22.25; 22.80;24.25; 24.66; 26.04; 26.44; 28.32; 28.96; 29.65; 30.22; 30.46; 30.78;32.17; 33.65; 34.49; 35.08; 35.33; 37.85; 38.48 as shown in FIG. 2.

In certain embodiments, the TG thermogram of a crystalline compound ofFormula (II) exhibits from 0.0 to 0.3% weight loss in the temperaturerange of 25 to 125° C. as shown in FIG. 3.

In certain embodiments, a crystalline compound of Formula (II) is notsolvated (e.g., the crystal lattice does not comprise molecules of asolvent). In certain alternative embodiments, a crystalline compound ofFormula (II) is solvated.

In certain embodiments, the invention relates to a method for thepreparation of a crystalline compound of Formula (II),

comprising (i) reacting a compound of Formula (III)

wherein X is any suitable counterion, with a compound of Formula (IV) inan organic solvent

(ii) preparing a solution of a compound of Formula (II) in the organicsolvent; (iii) bringing the solution to supersaturation to permitformation of crystals; and (iv) isolating the crystals to provide acrystalline compound of Formula (II), e.g., by filtering the crystals,by decanting, or by any other suitable separation technique.

In certain embodiments a compound of Formula (II) is not purified bychromatography prior to preparation of the solution in the organicsolvent.

In certain embodiments, preparation further comprises inducingcrystallization. In certain embodiments, preparation further compriseswashing the crystals, e.g., with a solvent or non-solvent fluid. Incertain embodiments, preparation further comprises drying, preferablyunder reduced pressure, such as under vacuum pressure.

In certain embodiments, X is a counterion selected from hydrobromide,hydrochloride, sulfate, phosphate, nitrate, acetate, trifluoroacetate,citrate, methanesulfonate, valerate, oleate, palmitate, stearate,laurate, benzoate, lactate, succinate, tosylate, malonate, maleate,fumarate, succinate, tartrate, mesylate, 2-hydroxyethanesulfonate, andthe like. (See, for example, Berge et al. (1977) “Pharmaceutical Salts”,J. Pharm. Sci. 66: 1-19.) In certain embodiments, X is selected fromtrifluoroacetate, methanesulfonate, toluenesulfonate, acetate, chloride,and bromide, preferably trifluoroacetate.

In certain embodiments, the organic solvent is selected fromacetonitrile, ethyl acetate, heptanes, hexanes, isopropyl acetate,methanol, methylethyl ketone, tetrahydrofuran, toluene, and water, orany combination thereof. In certain embodiments, the amorphous compoundof Formula (II) may be dissolved in an organic solvent selected fromacetonitrile, heptanes, hexanes, methanol, tetrahydrofuran, and toluene,or any combination thereof. In certain preferred embodiments, theorganic solvent is toluene, tetrahydrofuran, or acetonitrile, preferablyacetonitrile or toluene.

In certain embodiments, preparation further comprises washing thecrystals of Formula (II). In certain embodiments, washing the crystalscomprises washing with a liquid selected from anti-solvent,acetonitrile, heptanes, hexanes, methanol, tetrahydrofuran, toluene,water, or a combination thereof. In certain embodiments, the crystalsare washed with a combination of anti-solvent and the organic solvent.In certain embodiments, the anti-solvent is water, while in otherembodiments it is an alkane solvent, such as hexane or pentane, or anaromatic hydrocarbon solvent, such as benzene, toluene, or xylene.

In certain embodiments, preparation further comprises drying thecrystals of both of Formula (II), preferably under reduced pressure,such as under vacuum pressure.

In certain embodiments, the invention relates to a pharmaceuticalcomposition comprising a crystalline compound of Formula (II) and apharmaceutically acceptable carrier. In certain embodiments, thepharmaceutical composition is selected from tablets, capsules, andinjections.

Uses of Crystalline Tripeptide Epoxy Ketones

Orderly protein degradation is crucial to the maintenance of normal cellfunctions, and the proteasome is integral to the protein degradationprocess. The proteasome controls the levels of proteins that areimportant for cell-cycle progression and apoptosis in normal andmalignant cells; for example, cyclins, caspases, BCL2 and nF-kB(Kumatori et al., Proc. Natl. Acad. Sci. USA (1990) 87:7071-7075; Almondet al., Leukemia (2002) 16: 433-443). Thus, it is not surprising thatinhibiting proteasome activity can translate into therapies to treatvarious disease states, such as malignant, non-malignant and autoimmunediseases, depending on the cells involved.

Both in vitro and in vivo models have shown that malignant cells, ingeneral, are susceptible to proteasome inhibition. In fact, proteasomeinhibition has already been validated as a therapeutic strategy for thetreatment of multiple myeloma. This could be due, in part, to the highlyproliferative malignant cell's dependency on the proteasome system torapidly remove proteins (Rolfe et al., J. Mol. Med. (1997) 75:5-17;Adams, Nature (2004) 4: 349-360). Therefore, certain embodiments of theinvention relate to a method of treating a cancer, comprisingadministering to a subject in need of such treatment an effective amountof a proteasome inhibitor compound disclosed herein. As used herein, theterm “cancer” includes, but is not limited to, blood borne and solidtumors. Cancer refers to disease of blood, bone, organs, skin tissue andthe vascular system, including, but not limited to, cancers of thebladder, blood, bone, brain, breast, cervix, chest, colon, endometrium,esophagus, eye, head, kidney, liver, lung, lymph nodes, mouth, neck,ovaries, pancreas, prostate, rectum, renal, skin, stomach, testis,throat, and uterus. Specific cancers include, but are not limited to,leukemia (acute lymphocytic leukemia (ALL), acute myelogenous leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia(CML), hairy cell leukemia), mature B cell neoplasms (small lymphocyticlymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma(such as Waldenström's macroglobulinemia), splenic marginal zonelymphoma, plasma cell mycloma, plasmacytoma, monoclonal immunoglobulindeposition diseases, heavy chain diseases, extranodal marginal zone Bcell lymphoma (MALT lymphoma), nodal marginal zone B cell lymphoma(NMZL), follicular lymphoma, mantle cell lymphoma, diffuse B celllymphoma, mediastinal (thymic) large B cell lymphoma, intravascularlarge B cell lymphoma, primary effusion lymphoma and Burkittlymphoma/leukemia), mature T cell and natural killer (NK) cell neoplasms(T cell prolymphocytic leukemia, T cell large granular lymphocyticleukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma,extranodal NK/T cell lymphoma, enteropathy-type T cell lymphoma,hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosisfungoides (Sezary syndrome), primary cutaneous anaplastic large celllymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma,unspecified peripheral T cell lymphoma and anaplastic large celllymphoma), Hodgkin's lymphoma (nodular sclerosis, mixed celluarity,lymphocyte-rich, lymphocyte depleted or not depleted, nodularlymphocyte-predominant), myeloma (multiple myeloma, indolent myeloma,smoldering myeloma), chronic myeloproliferative disease,myelodysplastic/myeloproliferative disease, myelodysplastic syndromes,immunodeficiency-associated lymphoproliferative disorders, histiocyticand dendritic cell neoplasms, mastocytosis, chondrosarcoma, Ewingsarcoma, fibrosarcoma, malignant giant cell tumor, myeloma bone disease,osteosarcoma, breast cancer (hormone dependent, hormone independent),gynecological cancers (cervical, endometrial, fallopian tube,gestational trophoblastic disease, ovarian, peritoneal, uterine, vaginaland vulvar), basal cell carcinoma (BCC), squamous cell carcinoma (SCC),malignant melanoma, dermatofibrosarcoma protuberans, Merkel cellcarcinoma, Kaposi's sarcoma, astrocytoma, pilocytic astrocytoma,dysembryoplastic neuroepithelial tumor, oligodendrogliomas, ependymoma,glioblastoma multiforme, mixed gliomas, oligoastrocytomas,medulloblastoma, retinoblastoma, neuroblastoma, germinoma, teratoma,malignant mesothelioma (peritoneal mesothelioma, pericardialmesothelioma, pleural mesothelioma), gastro-entero-pancreatic orgastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid,pancreatic endocrine tumor (PET), colorectal adeno carcinoma, colorectalcarcinoma, aggressive neuroendocrine tumor, leiomyosarcoma, mucinousadenocarcinoma, Signet Ring cell adenocarcinoma, hepatocellularcarcinoma, cholangiocarcinoma, hepatoblastoma, hemangioma, hepaticadenoma, focal nodular hyperplasia (nodular regenerative hyperplasia,hamartoma), non-small cell lung carcinoma (NSCLC) (squamous cell lungcarcinoma, adenocarcinoma, large cell lung carcinoma), small cell lungcarcinoma, thyroid carcinoma, prostate cancer (hormone refractory,androgen independent, androgen dependent, hormone-insensitive), renalcell carcinoma, and soft tissue sarcomas (fibrosarcoma, malignantfibrous hystiocytoma, dermatofibrosarcoma, liposarcoma, rhabdomyosarcomaleiomyosarcoma, hemangiosarcoma, synovial sarcoma, malignant peripheralnerve sheath tumor/neurofibrosarcoma, extraskeletal osteosarcoma).

Many tumors of the haematopoietic and lymphoid tissues are characterizedby an increase in cell proliferation, or a particular type of cell. Thechronic myeloproliferative diseases (CMPDs) are clonal haematopoieticstem cell disorders characterized by proliferation in the bone marrow ofone or more of the myeloid lineages, resulting in increased numbers ofgranulocytes, red blood cells and/or platelets in the peripheral blood.As such, the use of proteasome inhibitors for the treatment of suchdiseases is attractive and being examined (Cilloni et al., Haematologica(2007) 92: 1124-1229). CMPD can include chronic myelogenous leukaemia,chronic neutrophilic leukaemia, chronic eosinophilic leukaemia,polycythaemia vera, chronic idiopathic myelofibrosis, essentialthrombocythaemia and unclassifiable chronic myeloproliferative disease.An aspect of the invention is the method of treating CMPD comprisingadministering to a subject in need of such treatment an effective amountof a proteasome inhibitor compound disclosed herein.

Myelodisplastic/myeloproliferative diseases, such as chronicmyelomonocytic leukaemia, atypical chronic myeloid leukemia, juvenilemyelomonocytic leukaemia and unclassifiablemyelodysplastic/myeloproliferative disease, are characterized byhypercellularity of the bone marrow due to proliferation in one or moreof the myeloid lineages. Inhibiting the proteasome with a compound orcomposition as described herein can serve to treat thesemyelodysplatic/myeloproliferative diseases by providing a subject inneed of such treatment an effective amount of the compound orcomposition.

Myelodysplastic syndromes (MDS) refer to a group of hematopoietic stemcell disorders characterized by dysplasia and ineffective haematopoiesisin one or more of the major myeloid cell lines. Targeting NF-kB with aproteasome inhibitor in these hematologic malignancies inducesapoptosis, thereby killing the malignant cell (Braun et al. Cell Deathand Differentiation (2006) 13:748-758). A further embodiment of theinvention is a method to treat MDS comprising administering to a subjectin need of such treatment an effective amount of a compound disclosedherein. MDS includes refractory anemia, refractory anemia with ringedsideroblasts, refractory cytopenia with multilineage dysplasia,refractory anemia with excess blasts, unclassifiable myelodysplasticsyndrome and myelodysplastic syndrome associated with isolated del(5q)chromosome abnormality.

Mastocytosis is a proliferation of mast cells and their subsequentaccumulation in one or more organ systems. Mastocytosis includes, but isnot limited to, cutaneous mastocytosis, indolent systemic mastocytosis(ISM), systemic mastocytosis with associated clonal haematologicalnon-mast-cell-lineage disease (SM-AHNMD), aggressive systemicmastocytosis (ASM), mast cell leukemia (MCL), mast cell sarcoma (MCS)and extracutaneous mastocytoma. Another embodiment of the invention is amethod to treat mastocytosis, comprising administering an effectiveamount of a compound or composition disclosed herein to a subjectdiagnosed with mastocytosis.

The proteasome regulates NF-κB, which in turn regulates genes involvedin the immune and inflammatory response. For example, NF-κB is requiredfor the expression of the immunoglobulin light chain κ gene, the IL-2receptor α-chain gene, the class I major histocompatibility complexgene, and a number of cytokine genes encoding, for example, IL-2, IL-6,granulocyte colony-stimulating factor, and IFN-β (Palombella et al.,Cell (1994) 78:773-785). Thus, in certain embodiments, the inventionrelates to methods of affecting the level of expression of IL-2, MHC-1,IL-6, TNFα, IFN-β or any of the other previously-mentioned proteins,each method comprising administering to a subject an effective amount ofa proteasome inhibitor compound or composition disclosed herein. Incertain embodiments, the invention includes a method of treating anautoimmune disease in a mammal comprising administering atherapeutically effective amount of a compound or composition describedherein. An “autoimmune disease” herein is a disease or disorder arisingfrom and directed against an individual's own tissues. Examples ofautoimmune diseases or disorders include, but are not limited to,inflammatory responses such as inflammatory skin diseases includingpsoriasis and dermatitis (e.g., atopic dermatitis); systemic sclerodermaand sclerosis; responses associated with inflammatory bowel disease(such as Crohn's disease and ulcerative colitis); respiratory distresssyndrome (including adult respiratory distress syndrome; ARDS);dermatitis; meningitis; encephalitis; uveitis; colitis;glomerulonephritis; allergic conditions such as eczema and asthma andother conditions involving infiltration of T cells and chronicinflammatory responses; atherosclerosis; leukocyte adhesion deficiency;rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetesmellitus (e.g., Type I diabetes mellitus or insulin dependent diabetesmellitis); multiple sclerosis; Reynaud's syndrome; autoimmunethyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenileonset diabetes; and immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes typically foundin tuberculosis, sarcoidosis, polymyositis, granulomatosis andvasculitis; pernicious anemia (Addison's disease); diseases involvingleukocyte diapedesis; central nervous system (CNS) inflammatorydisorder; multiple organ injury syndrome; hemolytic anemia (including,but not limited to cryoglobinemia or Coombs positive anemia); myastheniagravis; antigen-antibody complex mediated diseases; anti-glomerularbasement membrane disease; antiphospholipid syndrome; allergic neuritis;Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Beheet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

The immune system screens for autologous cells that are virallyinfected, have undergone oncogenic transformation, or present unfamiliarpeptides on their surface. Intracellular proteolysis generates smallpeptides for presentation to T-lymphocytes to induce MHC classI-mediated immune responses. Thus, in certain embodiments, the inventionrelates to a method of using the compound as an immunomodulatory agentfor inhibiting or altering antigen presentation in a cell, comprisingexposing the cell (or administering to a subject) to a compounddescribed herein. Specific embodiments include a method of treatinggraft or transplant-related diseases, such as graft-versus-host diseaseor host versus-graft disease in a mammal, comprising administering atherapeutically effective amount of a compound described herein. Theterm “graft” as used herein refers to biological material derived from adonor for transplantation into a recipient. Grafts include such diversematerial as, for example, isolated cells such as islet cells; tissuesuch as the amniotic membrane of a newborn, bone marrow, hematopoieticprecursor cells, and ocular tissue, such as corneal tissue; and organssuch as skin, heart, liver, spleen, pancreas, thyroid lobe, lung,kidney, tubular organs (e.g., intestine, blood vessels, or esophagus).The tubular organs can be used to replace damaged portions of esophagus,blood vessels, or bile duct. The skin grafts can be used not only forburns, but also as a dressing to damaged intestine or to close certaindefects such as diaphragmatic hernia. The graft is derived from anymammalian source, including human, whether from cadavers or livingdonors. In some cases, the donor and recipient is the same mammal.Preferably the graft is bone marrow or an organ such as heart and thedonor of the graft and the host are matched for HLA class II antigens.

Histiocytic and dendritic cell neoplasms are derived from phagocytes andaccessory cells, which have major roles in the processing andpresentation of antigens to lymphocytes. Depleting the proteasomecontent in dendritic cells has been shown to alter their antigen-inducedresponses (Chapatte et al. Cancer Res. (2006) 66:5461-5468). Thus,another embodiment of the invention comprises administering an effectiveamount of a compound or composition disclosed herein to a subject withhistiocytic or dendritic cell neoplasm. Histiocytic and dendritic cellneoplasms include histiocytic sarcoma, Langerhans cell histiocytosis,Langerhans cell sarcoma, interdigitating dendritic cell sarcoma/tumor,follicular dendritic cell sarcoma/tumor and non-specified dendritic cellsarcoma.

Inhibition of the proteasome has been shown to be beneficial to treatdiseases whereby a cell type is proliferating and immune disorders;thus, an embodiment of the invention includes the treatment oflymphoproliferative diseases (LPD) associated with primary immunedisorders (PID) comprising administering an effective amount of thedisclosed compound to a subject in need thereof. The most commonclinical settings of immunodeficiency associated with an increasedincidence of lymphoproliferative disorders, including B-cell and T-cellneoplasms and lymphomas, are primary immunodeficiency syndromes andother primary immune disorders, infection with the humanimmunodeficiency virus (HIV), iatrogenic immunosuppression in patientswho have received solid organ or bone marrow allografts, and iatrogenicimmunosuppression associated with methotrexate treatment. Other PIDscommonly associated with LPDs, but not limited to, are ataxiatelangiectasia (AT), Wiskott-Aldrich syndrome (WAS), common variableimmunodeficiency (CVID), severe combined immunodeficiency (SCID),X-linked lymphoproliferative disorder (XLP), Nijmegen breakage syndrome(NBS), hyper-IgM syndrome, and autoimmune lymphoproliferative syndrome(ALPS).

Additional embodiments of the invention relate to methods for affectingthe proteasome-dependent regulation of oncoproteins and methods oftreating or inhibiting cancer growth, each method comprising exposing acell (in vivo, e.g., in a subject, or in vitro) to the proteasomeinhibitor composition disclosed herein. HPV-16 and HPV-18-derived E6proteins stimulate ATP- and ubiquitin-dependent conjugation anddegradation of p53 in crude reticulocyte lysates. The recessive oncogenep53 has been shown to accumulate at the nonpermissive temperature in acell line with a mutated thermolabile E1. Elevated levels of p53 maylead to apoptosis. Examples of proto-oncoproteins degraded by theubiquitin system include c-Mos, c-Fos, and c-Jun. In certainembodiments, the invention relates to a method for treating p53-relatedapoptosis, comprising administering to a subject an effective amount ofa proteasome inhibitor composition disclosed herein.

Another aspect of the invention relates to the use of proteasomeinhibitor compositions disclosed herein for the treatment ofneurodegenerative diseases and conditions, including, but not limitedto, stroke, ischemic damage to the nervous system, neural trauma (e.g.,percussive brain damage, spinal cord injury, and traumatic damage to thenervous system), multiple sclerosis and other immune-mediatedneuropathies (e.g., Guillain-Barre syndrome and its variants, acutemotor axonal neuropathy, acute inflammatory demyelinatingpolyneuropathy, and Fisher Syndrome), HIV/AIDS dementia complex,axonomy, diabetic neuropathy, Parkinson's disease, Huntington's disease,multiple sclerosis, bacterial, parasitic, fungal, and viral meningitis,encephalitis, vascular dementia, multi-infarct dementia, Lewy bodydementia, frontal lobe dementia such as Pick's disease, subcorticaldementias (such as Huntington or progressive supranuclear palsy), focalcortical atrophy syndromes (such as primary aphasia), metabolic-toxicdementias (such as chronic hypothyroidism or B12 deficiency), anddementias caused by infections (such as syphilis or chronic meningitis).

Alzheimer's disease is characterized by extracellular deposits ofβ-amyloid protein (β-AP) in senile plaques and cerebral vessels. β-AP isa peptide fragment of 39 to 42 amino acids derived from an amyloidprotein precursor (APP). At least three isoforms of APP are known (695,751, and 770 amino acids). Alternative splicing of mRNA generates theisoforms; normal processing affects a portion of the β-AP sequence,thereby preventing the generation of β-AP. It is believed that abnormalprotein processing by the proteasome contributes to the abundance ofβ-AP in the Alzheimer brain. The APP-processing enzyme in rats containsabout ten different subunits (22 kDa-32 kDa). The 25 kDa subunit has anN-terminal sequence of X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which isidentical to the β-subunit of human macropain (Kojima, S. et al., Fed.Eur. Biochem. Soc., (1992) 304:57-60). The APP-processing enzyme cleavesat the Gln¹⁵-Lys¹⁶ bond; in the presence of calcium ion, the enzyme alsocleaves at the Met⁻¹-Asp¹ bond and the Asp¹-Ala² bond to release theextracellular domain of β-AP.

One aspect of the invention, therefore, relates to a method of treatingAlzheimer's disease, comprising administering to a subject an effectiveamount of a proteasome inhibitor compound or composition disclosedherein. Such treatment includes reducing the rate of β-AP processing,reducing the rate of β-AP plaque formation, reducing the rate of β-APgeneration, and reducing the clinical signs of Alzheimer's disease.

Fibrosis is the excessive and persistent formation of fibrous connectivetissue resulting from the hyperproliferative growth of fibroblasts andis associated with activation of the TGF-β signaling pathway. Fibrosisinvolves extensive deposition of extracellular matrix and can occurwithin virtually any tissue or across several different tissues.Normally, the level of intracellular signaling protein (Smad) thatactivates transcription of target genes upon TGF-β stimulation isregulated by proteasome activity (Xu et al., 2000). However, accelerateddegradation of the TGF-β signaling components has been observed infibrotic conditions, such as cystic fibrosis, injection fibrosis,endomyocardial fibrosis, idiopathic pulmonary fibrosis, myelofibrosis,retroperitoneal fibrosis, progressive massive fibrosis, nephrogenicsystemic fibrosis. Other conditions that are often associated withfibrosis include cirrhosis, diffuse parenchymal lung disease,post-vasectomy pain syndrome, tuberculosis, sickle-cell anemia andrheumatoid arthritis. An embodiment of the invention is the method oftreating a fibrotic or fibrotic-associated condition comprisingadministering an effective amount of the composition described herein toa subject in need of such treatment.

The treatment of burn victims is often hampered by fibrosis. Thus, incertain embodiments, the invention relates to the topical or systemicadministration of a subject inhibitor to treat burns. Wound closurefollowing surgery is often associated with disfiguring scars, which maybe prevented by inhibition of fibrosis. Thus, in certain embodiments,the invention relates to a method for the prevention or reduction ofscarring.

Overproduction of lipopolysaccharide (LPS)-induced cytokines such asTNFα is considered to be central to the processes associated with septicshock. Furthermore, it is generally accepted that the first step in theactivation of cells by LPS is the binding of LPS to specific membranereceptors. The α- and β-subunits of the 20S proteasome complex have beenidentified as LPS-binding proteins, suggesting that the LPS-inducedsignal transduction may be an important therapeutic target in thetreatment or prevention of sepsis (Qureshi, N. et al., J. Immun. (2003)171: 1515-1525). Therefore, in certain embodiments, the proteasomeinhibitor composition may be used for the inhibition of TNFα to preventand/or treat septic shock.

Ischemia and reperfusion injury results in hypoxia, a condition in whichthere is a deficiency of oxygen reaching the tissues of the body. Thiscondition causes increased degradation of Iκ-B α, thereby resulting inthe activation of NF-κB (Koong et al., 1994). It has been demonstratedthat the severity of injury resulting in hypoxia can be reduced with theadministration of a proteasome inhibitor (Gao et al., 2000; Bao et al.,2001; Pye et al., 2003). Therefore, certain embodiments of the inventionrelate to a method of treating an ischemic condition or reperfusioninjury comprising administering to a subject in need of such treatmentan effective amount of the proteasome inhibitor compound disclosedherein. Examples of such conditions or injuries include, but are notlimited to, acute coronary syndrome (vulnerable plaques), arterialocclusive disease (cardiac, cerebral, peripheral arterial and vascularocclusions), atherosclerosis (coronary sclerosis, coronary arterydisease), infarctions, heart failure, pancreatitis, myocardialhypertrophy, stenosis, and restenosis.

NF-κB also binds specifically to the HIV-enhancer/promoter. Whencompared to the Nef of mac239, the HIV regulatory protein Nef of pbj14differs by two amino acids in the region which controls protein kinasebinding. It is believed that the protein kinase signals thephosphorylation of IκB, triggering IκB degradation through theubiquitin-proteasome pathway. After degradation, NF-κB is released intothe nucleus, thus enhancing the transcription of HIV (Cohen, J.,Science, (1995) 267:960). In certain embodiments, the invention relatesto a method for inhibiting or reducing HIV infection in a subject, or amethod for decreasing the level of viral gene expression, each methodcomprising administering to the subject an effective amount of aproteasome inhibitor compound or composition disclosed herein.

Viral infections contribute to the pathology of many diseases. Heartconditions such as ongoing myocarditis and dilated cardiomyopathy havebeen linked to the coxsackievirus B3. In a comparative whole-genomemicroarray analyses of infected mouse hearts, specific proteasomesubunits were uniformly up-regulated in hearts of mice which developedchronic myocarditis (Szalay et al, Am J Pathol 168:1542-52, 2006). Someviruses utilize the ubiquitin-proteasome system in the viral entry stepwhere the virus is released from the endosome into the cytosol. Themouse hepatitis virus (MHV) belongs to the Coronaviridae family, whichalso includes the severe acute respiratory syndrome (SARS) coronavirus.Yu and Lai (J Virol 79:644-648, 2005) demonstrated that treatment ofcells infected with MHV with a proteasome inhibitor resulted in adecrease in viral replication, correlating with reduced viral titer ascompared to that of untreated cells. The human hepatitis B virus (HBV),a member of the Hepadnaviridae virus family, likewise requires virallyencoded envelop proteins to propagate. Inhibiting the proteasomedegradation pathway causes a significant reduction in the amount ofsecreted envelope proteins (Simsek et al, J Virol 79:12914-12920, 2005).In addition to HBV, other hepatitis viruses (A, C, D and E) may alsoutilize the ubiquitin-proteasome degradation pathway for secretion,morphogenesis and pathogenesis. Accordingly, in certain embodiments, theinvention relates to a method for treating viral infection, such as SARSor hepatitis A, B, C, D and E, comprising contacting a cell with (oradministering to a subject) an effective amount of a compound orcomposition disclosed herein.

In certain embodiments, the disclosed compositions may be useful for thetreatment of a parasitic infection, such as infections caused byprotozoan parasites. The proteasome of these parasites is considered tobe involved primarily in cell differentiation and replication activities(Paugam et al., Trends Parasitol. 2003, 19(2): 55-59). Furthermore,entamoeba species have been shown to lose encystation capacity whenexposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res.1997, 28, Spec No: 139-140). In certain such embodiments, theadministrative protocols for the proteasome inhibitor compositions areuseful for the treatment of parasitic infections in humans caused by aprotozoan parasite selected from Plasmodium sps. (including P.falciparum, P. vivax, P. malariae, and P. ovale, which cause malaria),Trypanosoma sps. (including T. cruzi, which causes Chagas' disease, andT. brucei which causes African sleeping sickness), Leishmania sps.(including L. amazonesis, L. donovani, L. infantum, L. mexicana, etc.),Pneumocystis carinii (a protozoan known to cause pneumonia in AIDS andother immunosuppressed patients), Toxoplasma gondii, Entamoebahistolytica, Entamoeba invadens, and Giardia lamblia. In certainembodiments, the disclosed proteasome inhibitor compositions are usefulfor the treatment of parasitic infections in animals and livestockcaused by a protozoan parasite selected from Plasmodium hermani,Cryptosporidium sps., Echinococcus granulosus, Eimeria tenella,Sarcocystis neurona, and Neurospora crassa. Other compounds that act asproteasome inhibitors in the treatment of parasitic diseases aredescribed in WO 98/10779, which is incorporated herein in its entirety.

In certain embodiments, the proteasome inhibitor compositions inhibitproteasome activity in a parasite without recovery in red blood cellsand white blood cells. In certain such embodiments, the long half-lifeof blood cells may provide prolonged protection with regard to therapyagainst recurring exposures to parasites. In certain embodiments, theproteasome inhibitor compositions may provide prolonged protection withregard to chemoprophylaxis against future infection.

Prokaryotes have an equivalent to the eukaryote 20S proteasome particle.Although the subunit composition of the prokaryote 20S particle issimpler than that of eukaryotes, it has the ability to hydrolyze peptidebonds in a similar manner. For example, the nucleophilic attack on thepeptide bond occurs through the threonine residue on the N-terminus ofthe β-subunits. Thus, an embodiment of this invention relates to amethod of treating prokaryotic infections, comprising administering to asubject an effective amount of a proteasome inhibitor compound orcomposition disclosed herein. Prokaryotic infections may includediseases caused by either mycobacteria (such as tuberculosis, leprosy orBuruli ulcer) or archaebacteria.

It has also been demonstrated that inhibitors that bind to the 20Sproteasome stimulate bone formation in bone organ cultures. Furthermore,when such inhibitors have been administered systemically to mice,certain proteasome inhibitors increased bone volume and bone formationrates over 70% (Garrett, I. R. et al., J. Clin. Invest. (2003) 111:1771-1782), therefore suggesting that the ubiquitin-proteasome machineryregulates osteoblast differentiation and bone formation. Therefore, adisclosed proteasome inhibitor compound or composition may be useful inthe treatment and/or prevention of diseases associated with bone loss,such as osteoporosis.

Thus, in certain embodiments, the invention relates to a method fortreating a disease or condition selected from cancer, autoimmunedisease, graft or transplant-related condition, neurodegenerativedisease, fibrotic-associated condition, ischemic-related conditions,infection (viral, parasitic or prokaryotic) and diseases associated withbone loss, comprising administering a compound or composition asdisclosed herein.

Administration of Crystalline Tripeptide Epoxy Ketones

Compounds prepared as described herein can be administered in variousforms, depending on the disorder to be treated and the age, condition,and body weight of the patient, as is well known in the art. Forexample, where the compounds are to be administered orally, they may beformulated as tablets, capsules, granules, powders, or syrups; or forparenteral administration, they may be formulated as injections(intravenous, intramuscular, or subcutaneous), drop infusionpreparations, or suppositories. For application by the ophthalmic mucousmembrane route, they may be formulated as eye drops or eye ointments.These formulations can be prepared by conventional means, and ifdesired, the active ingredient may be mixed with any conventionaladditive or excipient, such as a binder, a disintegrating agent, alubricant, a corrigent, a solubilizing agent, a suspension aid, anemulsifying agent, a coating agent, a cyclodextrin, and/or a buffer.Although the dosage will vary depending on the symptoms, age and bodyweight of the patient, the nature and severity of the disorder to betreated or prevented, the route of administration and the form of thedrug, in general, a daily dosage of from 0.01 to 2000 mg of the compoundis recommended for an adult human patient, and this may be administeredin a single dose or in divided doses. The amount of active ingredientwhich can be combined with a carrier material to produce a single dosageform will generally be that amount of the compound which produces atherapeutic effect.

The precise time of administration and/or amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage, and type of medication),route of administration, etc. However, the above guidelines can be usedas the basis for fine-tuning the treatment, e.g., determining theoptimum time and/or amount of administration, which will require no morethan routine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose, and sucrose; (2) starches, such as corn starch, potatostarch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6)gelatin; (7) talc; (8) excipients, such as cocoa butter and suppositorywaxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such aspropylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol,and polyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations. In certainembodiments, pharmaceutical compositions of the present invention arenon-pyrogenic, i.e., do not induce significant temperature elevationswhen administered to a patient.

The term “pharmaceutically acceptable salt” refers to the relativelynon-toxic, inorganic and organic acid addition salts of theinhibitor(s). These salts can be prepared in situ during the finalisolation and purification of the inhibitor(s), or by separatelyreacting a purified inhibitor(s) in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, laurylsulphonate salts, and amino acidsalts, and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19.)

In other cases, the inhibitors useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic inorganicand organic base addition salts of an inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the inhibitor(s), or by separately reacting the purified inhibitor(s)in its free acid form with a suitable base, such as the hydroxide,carbonate, or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like(see, for example, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring, and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like;(2) oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert matrix, such as gelatin and glycerin, orsucrose and acacia) and/or as mouthwashes, and the like, each containinga predetermined amount of an inhibitor(s) as an active ingredient. Acomposition may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), the active ingredient may bemixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, cyclodextrins, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets, and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols, andthe like. In certain embodiments the crystalline tripeptide epoxyketoneis administered to a mammal as a capsule. In another embodiment, thecrystalline tripeptide epoxyketone is a compound of formula (I). In amore preferred embodiment, the crystalline tripeptide epoxyketone is acompound of formula (II).

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered inhibitor(s)moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills,and granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes, and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents, and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor, and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active inhibitor(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more inhibitor(s)with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, which is solid at room temperature, butliquid at body temperature and, therefore, will melt in the rectum orvaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of aninhibitor(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, and inhalants. The active componentmay be mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams, and gels may contain, in addition toinhibitor(s), excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an inhibitor(s),excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

The inhibitor(s) can be alternatively administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation, orsolid particles containing the composition. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular composition,but typically include nonionic surfactants (Tweens, Pluronics, sorbitanesters, lecithin, Cremophors), pharmaceutically acceptable co-solventssuch as polyethylene glycol, innocuous proteins like serum albumin,oleic acid, amino acids such as glycine, buffers, salts, sugars, orsugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of an inhibitor(s) to the body. Such dosage forms can be madeby dissolving or dispersing the agent in the proper medium. Absorptionenhancers can also be used to increase the flux of the inhibitor(s)across the skin. The rate of such flux can be controlled by eitherproviding a rate controlling membrane or dispersing the inhibitor(s) ina polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more inhibitors(s) in combination withone or more pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include tonicity-adjusting agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. For example, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming microcapsule matrices ofinhibitor(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are, of course, given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection, and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a ligand, drug, or other materialother than directly into the central nervous system, such that it entersthe patient's system and thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These inhibitors(s) may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally, and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the inhibitor(s),which may be used in a suitable hydrated form, and/or the pharmaceuticalcompositions of the present invention, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The concentration of a disclosed compound in a pharmaceuticallyacceptable mixture will vary depending on several factors, including thedosage of the compound to be administered, the pharmacokineticcharacteristics of the compound(s) employed, and the route ofadministration. In general, the compositions of this invention may beprovided in an aqueous solution containing about 0.1-10% w/v of acompound disclosed herein, among other substances, for parenteraladministration. Typical dose ranges are from about 0.01 to about 50mg/kg of body weight per day, given in 1-4 divided doses. Each divideddose may contain the same or different compounds of the invention. Thedosage will be an effective amount depending on several factorsincluding the overall health of a patient, and the formulation and routeof administration of the selected compound(s).

Definitions

The term “C_(x-y)alkyl” refers to substituted or unsubstituted saturatedhydrocarbon groups, including straight-chain alkyl and branched-chainalkyl groups that contain from x to y carbons in the chain, includinghaloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.C₀alkyl indicates a hydrogen where the group is in a terminal position,a bond if internal. The terms “C_(2-y)alkenyl” and “C_(2-y)alkynyl”refer to substituted or unsubstituted unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxy.

The term “C₁₋₆alkoxyalkyl” refers to a C₁₋₆alkyl group substituted withan alkoxy group, thereby forming an ether.

The term “C₁₋₆aralkyl”, as used herein, refers to a C₁₋₆alkyl groupsubstituted with an aryl group.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by the general formulae:

wherein R⁹, R¹⁰ and R^(10′) each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R⁸, or R⁹ and R¹⁰ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R⁸ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger from 1 to 8. In preferred embodiments, only one of R⁹ or R¹⁰ canbe a carbonyl, e.g., R⁹, R¹⁰, and the nitrogen together do not form animide. In even more preferred embodiments, R⁹ and R¹⁰ (and optionallyR^(10′)) each independently represent a hydrogen, an alkyl, an alkenyl,or —(CH₂)_(m)—R⁸. In certain embodiments, the amino group is basic,meaning the protonated form has a pK_(a)≧7.00.

The terms “amide” and “amido” are art-recognized as an amino-substitutedcarbonyl and includes a moiety that can be represented by the generalformula:

wherein R⁹, R¹⁰ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsubstituted or unsubstituted single-ring aromatic groups in which eachatom of the ring is carbon. The term “aryl” also includes polycyclicring systems having two or more cyclic rings in which two or morecarbons are common to two adjoining rings wherein at least one of therings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline,and the like.

The terms “carbocycle” and “carbocyclyl”, as used herein, refer to anon-aromatic substituted or unsubstituted ring in which each atom of thering is carbon. The terms “carbocycle” and “carbocyclyl” also includepolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is carbocyclic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R¹¹represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R⁸ or apharmaceutically acceptable salt, R^(11′) represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R⁸, where m and R⁸ are as defined above.Where X is an oxygen and R¹¹ or R^(11′) is not hydrogen, the formularepresents an “ester”. Where X is an oxygen, and R¹¹ is a hydrogen, theformula represents a “carboxylic acid”.

The terms “heteroaryl” includes substituted or unsubstituted aromatic 5-to 7-membered ring structures, more preferably 5- to 6-membered rings,whose ring structures include one to four heteroatoms. The term“heteroaryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings wherein at least one of the rings is heteroaromatic, e.g., theother cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, forexample, pyrrole, furan, thiophene, imidazole, isoxazole, oxazole,thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,phosphorus, and sulfur.

The terms “heterocyclyl” or “heterocyclic group” refer to substituted orunsubstituted non-aromatic 3- to 10-membered ring structures, morepreferably 3- to 7-membered rings, whose ring structures include one tofour heteroatoms. The term terms “heterocyclyl” or “heterocyclic group”also include polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings wherein atleast one of the rings is heterocyclic, e.g., the other cyclic rings canbe cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heterocyclyl groups include, for example,tetrahydrofuran, piperidine, piperazine, pyrrolidine, morpholine,lactones, lactams, and the like.

The term “C₁₋₆heterocycloalkyl”, as used herein, refers to a C₁₋₆alkylgroup substituted with a heterocyclyl group.

The term “C₁₋₆hydroxyalkyl” refers to a C₁₋₆alkyl group substituted witha hydroxy group.

As used herein, the term “inhibitor” is meant to describe a compoundthat blocks or reduces an activity of an enzyme (for example, inhibitionof proteolytic cleavage of standard fluorogenic peptide substrates suchas suc-LLVY-AMC, Box-LLR-AMC and Z-LLE-AMC, inhibition of variouscatalytic activities of the 20S proteasome). An inhibitor can act withcompetitive, uncompetitive, or noncompetitive inhibition. An inhibitorcan bind reversibly or irreversibly, and therefore the term includescompounds that are suicide substrates of an enzyme. An inhibitor canmodify one or more sites on or near the active site of the enzyme, or itcan cause a conformational change elsewhere on the enzyme.

As used herein, the term “orally bioavailable” is meant to describe acompound administered to a mouse at 40 mg/kg or less, 20 mg/kg or less,or even 10 mg/kg or less, wherein one hour after oral administrationsuch a compound shows at least about 50%, at least about 75% or even atleast about 90% inhibition of proteasome CT-L activity in the blood.

As used herein, the term “peptide” includes not only standard amidelinkage with standard α-substituents, but commonly utilizedpeptidomimetics, other modified linkages, non-naturally occurring sidechains, and side chain modifications, as detailed below.

The terms “polycyclyl” or “polycyclic” refer to two or more rings (e.g.,cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Each of the rings of thepolycycle can be substituted or unsubstituted.

The term “proteasome” as used herein is meant to include immuno- andconstitutive proteasomes.

The term “substantially pure” as used herein, refers to a crystallinepolymorph that is greater than 90% pure, meaning that contains less than10% of any other compound, including the corresponding amorphouscompound. Preferably, the crystalline polymorph is greater than 95%pure, or even greater than 98% pure.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “prodrug” encompasses compounds that, under physiologicalconditions, are converted into therapeutically active agents. A commonmethod for making a prodrug is to include selected moieties that arehydrolyzed under physiological conditions to reveal the desiredmolecule. In other embodiments, the prodrug is converted by an enzymaticactivity of the host animal.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “proteasome” as used herein is meant to include immuno- andconstitutive proteasomes.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include, for example, a halogen, ahydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl,or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, aphosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro,an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

The term “thioether” refers to an alkyl group, as defined above, havinga sulfur moiety attached thereto. In preferred embodiments, the“thioether” is represented by —S-alkyl. Representative thioether groupsinclude methylthio, ethylthio, and the like.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition.

EXEMPLIFICATION Example 1 Synthesis of Compound 1

Synthesis of (A)

To a 0° C. solution of N-Boc serine (methyl ether) (43.8 g, 200 mmol),triethylamine (26.5 g, 260 mmol) and 4-(dimethylamino)pyridine indichloromethane (1.2 L) was added a solution of benzyl chloroformate (41g, 240 mmol) in dichloromethane (250 mL) over 30 minutes. The resultingmixture was stirred at the same temperature for another 3 hours.Saturated aqueous sodium bicarbonate (200 mL) was added and organiclayer was separated, the residual mixture was extracted withdichloromethane (2×400 mL). The combined organic layers were washed withsaturated aqueous sodium bicarbonate (200 mL) and brine (200 mL), driedover sodium sulfate and filtered through Celite-545. The solvents wereremoved under reduced pressure and residue was purified by flashchromatography (silica gel, hexane and ethyl acetate). Compound (A) (54g) was isolated and characterized by LC/MS (LRMS(MH) m/z: 310.16).

Synthesis of (B)

To a 0° C. solution of Compound (A) (54 g) in dichloromethane (200 mL)was added trifluoroacetic acid (200 mL) over 10 minutes, and theresulting mixture was stirred at the same temperature for another 3hours. The solvents were removed under reduced pressure and the residuewas placed under high vacuum overnight giving the TFA salt of Compound(B), which was characterized by LC/MS (LRMS (MH) m/z: 210.11).

Synthesis of (C)

To a 0° C. solution of Compound (B) (43.8 g, 200 mmol), N-Boc serine(methyl ether) (36.7 g, 167 mmol), HOBT (27 g, 200 mmol) and HBTU (71.4g, 200 mmol) in tetrahydrofuran (1.2 L) was added a solution ofN,N-diethylisopropylamine (75 g, 600 mmol) in tetrahydrofuran (250 mL)over 10 minutes, and the pH of the resulting mixture was ˜8. The mixturewas stirred at room temperature for another 5 hours. Most of the solventwere removed under reduced pressure at room temperature and diluted withsaturated aqueous sodium bicarbonate (400 mL). Then it was extractedwith ethyl acetate (3×400 mL), washed with sodium bicarbonate (100 mL)and brine (100 mL). The combined organic layers were dried over sodiumsulfate and filtered through Celite-545. The solvents were removed underreduced pressure and residue was purified by flash chromatography(silica gel, hexane and ethyl acetate). Compound (C) (65 g) was isolatedand characterized by LC/MS (LRMS (MH) m/z: 411.21).

Synthesis of (D)

To a 0° C. solution of Compound (C) (18 g) in dichloromethane (100 mL)was added trifluoroacetic acid (80 mL) over 5 minutes, and the resultingmixture was stirred at the same temperature for another 3 hours. Thesolvents were removed under reduced pressure and the residue was placedunder high vacuum overnight giving the TFA salt of intermediate (D),which was characterized by LC/MS (LRMS (MH) m/z: 311.15).

Synthesis of (E)

To a 0° C. solution of ethyl 2-methyl-thiazole-5-carboxylate (15 g, 88mmol) in tetrahydrofuran (50 mL) was added aqueous sodium hydroxidesolution (5 N, 50 mL) over 10 minutes, and the resulting solution wasstirred at room temperature for another 2 hours. It was then acidifiedwith hydrochloric acid (2 N) to pH=1 and extracted with tetrahydrofuran(3×100 mL). The combined organic layers were washed with brine (30 mL)and dried over sodium sulfate. Most of the solvents were removed underreduced pressure and the residue was lyophilized to afford Compound (E)(14 g).

Synthesis of (F)

To a 0° C. solution of Compound (D) (41 mmol) and2-methyl-thiazole-5-carboxylic acid (E) (6.0 g, 42 mmol), HOBT (7.9 g,50 mmol) and HBTU (18.0 g, 50 mmol) in tetrahydrofuran (800 mL) wasadded a solution of N,N-diethylisopropylamine (˜50 g) in tetrahydrofuran(200 mL) over 5 minutes until its pH reached approximately 8.5. Theresulting mixture was stirred at same temperature overnight. It was thenquenched with saturated aqueous sodium bicarbonate solution (200 mL),and most of the solvents were removed under reduced pressure. Theresidual mixture was extracted with ethyl acetate (3×400 mL). Thecombined organic layers were washed with saturated aqueous sodiumbicarbonate (200 mL) and brine (100 mL), dried over sodium sulfate andfiltered through Celite-545. The solvents were removed under reducedpressure and residue was purified by flash chromatography (silica gel,ethyl acetate with 2% methanol). Compound (F) (17.1 g) was isolated andcharacterized by LC/MS (LRMS (MH) m/z: 436.15).

Synthesis of (G)

To a solution of Compound (F) (17.1 g, 95 mmol) in methanol (300 mL) wasadded 10% Pd/C (3 g). The resulting mixture was allowed to stir under 1atmosphere of hydrogen for 48 hours. The mixture was filtered throughCelite 545 and the filter cake was washed with methanol (˜200 mL). Theorganic layers were concentrated under reduced pressure and placed underhigh vacuum to yield Compound (G), which was characterized by LC/MS(LRMS (MH) m/z: 346.1).

Synthesis of (H)

N-Boc phenylalanine-ketoepoxide (140 mg, 0.46 mmol) was diluted with DCM(2 mL) and cooled to 0° C. To this solution was added trifluoroaceticacid (6 mL). The cooling bath was removed and the reaction stirred for 1hour at which time TLC showed complete consumption of starting material.The resulting solution was concentrated under reduced pressure andplaced under high vacuum to yield TFA salt of Compound (H).

Synthesis of Compound 1

To a 0° C. solution of aforementioned Compounds (H) (131 mg, 0.38 mmol)and (J) (0.46 mmol), HOBT (75 mg, 0.48 mmol) and HBTU (171 mg, 0.48mmol) in tetrahydrofuran (20 mL) and N,N-dimethylformamide (10 mL) wasadded N,N-diethylisopropylamine (1 mL) dropwise. The mixture was stirredat the same temperature for another 5 hours. It was then quenched withsaturated aqueous sodium bicarbonate solution (20 mL), and most of thesolvents were removed under reduced pressure. The residual mixture wasthen extracted with ethyl acetate (3×40 mL). The combined organic layerswere washed with saturated aqueous sodium bicarbonate (20 mL) and brine(10 mL), dried over sodium sulfate and filtered through Celite-545. Thesolvents were removed under reduced pressure and residue was purified byHPLC (0.02 M aqueous ammonium acetate and acetonitrile (66/34) to affordCompound 1 (92 mg), which was lyophilized and characterized by LC/MS(LRMS (MH) m/z: 533.2).

Example 2

Amorphous Compound 1 (50 mg) was dissolved in acetonitrile (1 mL), thendeionized water (2 mL) was added, and the solution brought tosupersaturation by slowly evaporating off 1 mL over about 1-2 weeks. Theresulting crystals were filtered, washed with 1 mL 1:2acetonitrile-water, and dried under vacuum for 12 hours to provide acrystalline polymorph of Compound 1 (25 mg) with a melting point of 148°C. The characteristic DSC curve of the sample is shown in FIG. 1 asrecorded on a TA Instruments Differential Scanning calorimeter 2920 at aheating rate of 10° C./minute.

Example 3

Amorphous Compound 1 (611 mg) was dissolved in tetrahydrofuran (5 mL),followed by addition of hexanes (5 mL) and the solution was seeded withcrystalline polymorph Compound 1 as prepared in Example 2, and thesolution brought to supersaturation by slowly evaporating off 5 mL overabout 17 hours. The resulting crystals were filtered, washed with 1 mL1:1 tetrahydrofuran-hexanes, and dried under vacuum for 12 hours toprovide a crystalline polymorph of Compound 1 (150 mg) with a meltingpoint of 147° C.

Example 4

Amorphous Compound 1 (176 mg) was dissolved in tetrahydrofuran (5 mL),then toluene (25 mL) was added. The solution was seeded with crystallinepolymorph Compound 1 as prepared in Example 2, and the solution wasbrought to supersaturation by slowly evaporating off 20 mL over about 2days. The resulting crystals were filtered, washed with 15 mL toluene,and dried under vacuum for 12 hours to provide a crystalline polymorphof Compound 1 (88 mg) with a melting point of 149° C.

Example 5

Amorphous Compound 1 (312 mg) was dissolved in toluene (50 mL), heatedto about 100° C. to complete dissolution, then hexanes (50 mL) wereadded and the solution was seeded with crystalline polymorph Compound 1as prepared in Example 2, and the solution brought to supersaturation byslowly evaporating off 60 mL over about 2 days. The resulting crystalswere filtered, washed with 10 mL toluene, and dried under vacuum for 12hours to provide a crystalline polymorph of Compound 1 (156 mg) with amelting point of 149° C.

Example 6

Amorphous Compound 1 (1.4 g) was dissolved in toluene (25 mL), heated toabout 50° C. to complete dissolution, then brought to supersaturation bycooling to 22° C. and allowing the compound to crystallize for 12 hours.The resulting crystals were filtered, washed with 5 mL hexanes, anddried under vacuum for 12 hours to provide a crystalline polymorph ofCompound 1 (0.94 g) with a melting point of 149° C.

Example 7 Synthesis of Compound 1

Synthesis of (H)

N-Boc phenylalanine-ketoepoxide (1.0 equivalent) was dissolved in DCM (3L/kg of N-Boc phenylalanine-ketoepoxide) in a 3-neck round bottom flaskunder inert atmosphere and the solution was cooled in ice bath. Then,TFA (5.0 equivalents) was added at a rate to maintain the internaltemperature below 10° C. The reaction mixture was then warmed toapproximately 20° C. and stirred for 1 to 3 hours. MTBE (3.6 L/kg ofN-Boc phenylalanine-ketoepoxide) was then added to the reaction mixturewhile maintaining mixture temperature below 25° C. Heptane (26.4 L/kg ofN-Boc phenylalanine-ketoepoxide) was then added the reaction was cooledto between −5 and 0° C. for 2 to 3 hours to allow crystallization ofCompound (H). The white solid was filtered and rinsed with heptane (3L/kg of N-Boc phenylalanine-ketoepoxide). The white solid was then undervacuum for 12 hours at 22° C. Yield obtained was 86%, with HPLC purity99.4%.

Synthesis of Compound 1

Compound (H) (1.2 equivalents), Compound (G) (1.0 equivalent), HBTU (1.2equivalents), HOBT (1.2 equivalents) and N-methyl pyrrolidinone (8 L/kgof Compound (G)) were added to a dry flask under inert atmosphere, andthe mixture was stirred at 23° C. to complete dissolution. The reactionwas then cooled to between −5 and 0° C., and diisopropylethylamine (2.1equivalents) was added over 15 minutes, while maintaining an internalreaction temperature of less than 0° C. The reaction mixture was stirredat 0° C. for 12 hours.

Crude Compound 1 was precipitated by pouring the reaction mixture onto8% sodium bicarbonate (40 L/kg of Compound (G)) and the suspension ofcrude Compound 1 was stirred for 12 hours at 20 to 25° C., followed bystirring at 0 to 5° C. for 1 hour. The white solid was filtered andrinsed with water (5 L/kg of Compound (G)). The white solid was thenreslurried in water (15 L/kg) for 3 hours at 20 to 25° C., filtered andrinsed with water (5 L/kg of Compound (G)) and isopropyl acetate (2×2L/kg of Compound (G)). The white solid was dried under vacuum at 45° C.to constant weight. Yield of crude Compound 1 was 65%, with HPLC purityof 97.2%.

Crude Compound 1 was completely dissolved in isopropyl acetate (20 L/kgof crude Compound 1) by stirring and heating at 85° C. The solution wasthen hot filtered to remove any particulate mater and the solution wasre-heated to 85° C. to provide clear solution. The clear solution wasallowed to cool at 10° C. per hour to 65° C. before adding seedcrystals. The solution was allowed to cool at 10° C. per hour to 20° C.,when substantial crystallization of Compound 1 occurred. The suspensionwas stirred at 20° C. for 6 hours, followed by stirring at 0 to 5° C.for a minimum of 2 hours and filtration and rinsing with isopropylacetate (1 L/kg of crude Compound 1). The purified Compound 1 was driedunder vacuum at 45° C. for a minimum of 24 hours to constant weight.Yield of Compound 1 was 87%, with HPLC purity 97.2%.

Example 8 Synthesis of Compound 1

Compound (H) (1.1 equivalents), Compound (G) (1.0 equivalent), HBTU (1.5equivalents), HOBT (1.5 equivalents) and DMF (8 L/kg of Compound (G))were added to a dry flask under inert atmosphere, and the mixture wasstirred at 23° C. to complete dissolution. The reaction was then cooledto between −5 and 0° C., and diisopropylethylamine (2.1 equivalents) wasadded over 15 minutes, while maintaining an internal reactiontemperature of less than 0° C. The reaction mixture was then stirred at0° C. for 3 hours.

The reaction mixture was quenched by addition of pre-chilled saturatedsodium bicarbonate (94 L/kg of Compound (G)), while maintaining internaltemperature of less 10° C. The content was then transferred to aseparatory funnel. The mixture was extracted with ethyl acetate (24 L/kgof Compound (G)), and the organic layer was washed with saturated sodiumbicarbonate (12 L/kg of Compound (G)) and with saturated sodium chloride(12 L/kg of Compound (G).

The organic layer was concentrated under reduced pressure with a bathtemperature of less than 30° C. to 15 L/kg of Compound (G), followed byco-distillation with isopropyl acetate (2×24 L/kg of PR-022). Finalvolume was adjusted to 82 L/kg of Compound (G) with isopropyl acetatebefore heating to 60° C. to obtain a clear solution. The clear solutionmixture was allowed to cool to 50° C. before adding seed crystals. Thesolution was allowed to cool to 20° C., when substantial crystallizationof Compound 1 had occurred. The suspension was stirred at 0° C. for 12hours before filtration and rinsing with isopropyl acetate (2 L/kg ofCompound 1). Compound 1 was dried under vacuum at 20° C. for 12 hours toconstant weight. Yield of Compound 1 was 48%, with HPLC purity of 97.4%.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds and methods of use thereof described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims.

All of the above-cited references and publications are herebyincorporated by reference.

We claim:
 1. A method for treating a cancer in a patient, the methodcomprising administering to the patient a pharmaceutical compositioncomprising a therapeutically effective amount of a crystalline compoundhaving a structure of Formula (II):

wherein the compound has 2θ values 8.94; 9.39; 9.76; 10.60; 11.09;12.74; 15.27; 17.74; 18.96; 20.58; 20.88; 21.58; 21.78; 22.25; 22.80;24.25; 24.66; 26.04; 26.44; 28.32; 28.96; 29.65; 30.22; 30.46; 30.78;32.17; 33.65; 34.49; 35.08; 35.33; 37.85; and 38.48; and apharmaceutically acceptable carrier, wherein the cancer is selected fromthe group consisting of leukemia, mature B cell neoplasm, mature T celland natural killer cell neoplasm, Hodgkin's lymphoma, myeloma,pancreatic cancer, bladder cancer, bone cancer, breast cancer, cervicalcancer, cancer of the chest, colon cancer, endometrial cancer,esophageal cancer, cancer of the eye, head and neck cancer, kidneycancer, liver cancer, lung cancer, lymphatic cancer, mouth cancer,ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renalcancer, skin cancer, stomach cancer, testicular cancer, throat cancer,and uterine cancer.
 2. The method of claim 1, wherein the myeloma ismultiple myeloma.
 3. The method of claim 1, wherein the pharmaceuticalcomposition is a tablet, capsule, granule, powder, or dragee.
 4. Themethod of claim 1, wherein the pharmaceutical composition isadministered orally.
 5. A method for treating an autoimmune disease in apatient, the method comprising administering to the patient apharmaceutical composition comprising a therapeutically effective amountof a crystalline compound having a structure of Formula (II):

wherein the compound has 2θ values 8.94; 9.39; 9.76; 10.60; 11.09;12.74; 15.27; 17.74; 18.96; 20.58; 20.88; 21.58; 21.78; 22.25; 22.80;24.25; 24.66; 26.04; 26.44; 28.32; 28.96; 29.65; 30.22; 30.46; 30.78;32.17; 33.65; 34.49; 35.08; 35.33; 37.85; and 38.48; and apharmaceutically acceptable carrier, wherein the autoimmune disease isselected from the group consisting of: multiple sclerosis; rheumatoidarthritis; lupus; systemic lupus erythematosus (SLE); Reynaud'ssyndrome; autoimmune thyroiditis; Sjogren's syndrome; juvenile onsetdiabetes; an immune response associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes typically foundin tuberculosis, sarcoidosis, polymyositis, granulomatosis andvasculitis; pernicious anemia (Addison's disease); a disease involvingleukocyte diapedesis; multiple organ injury syndrome; hemolytic anemia;myasthenia gravis; antigen-antibody complex mediated disease;anti-glomerular basement membrane disease; antiphospholipid syndrome;Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Beheet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP); and autoimmune thrombocytopenia.
 6. Themethod of claim 5, wherein the autoimmune disease is selected from thegroup consisting of: lupus, rheumatoid arthritis, and anantigen-antibody complex mediated disease.
 7. A method for treatinggraft-versus-host disease in a patient, the method comprisingadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of a crystalline compound having astructure of Formula (II):

wherein the compound has 2θ values 8.94; 9.39; 9.76; 10.60; 11.09;12.74; 15.27; 17.74; 18.96; 20.58; 20.88; 21.58; 21.78; 22.25; 22.80;24.25; 24.66; 26.04; 26.44; 28.32; 28.96; 29.65; 30.22; 30.46; 30.78;32.17; 33.65; 34.49; 35.08; 35.33; 37.85; and 38.48; and apharmaceutically acceptable carrier.
 8. A method for treating aneurodegenerative disease in a patient, the method comprisingadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of a crystalline compound having astructure of Formula (II):

wherein the compound has 2θ values 8.94; 9.39; 9.76; 10.60; 11.09;12.74; 15.27; 17.74; 18.96; 20.58; 20.88; 21.58; 21.78; 22.25; 22.80;24.25; 24.66; 26.04; 26.44; 28.32; 28.96; 29.65; 30.22; 30.46; 30.78;32.17; 33.65; 34.49; 35.08; 35.33; 37.85; and 38.48; and apharmaceutically acceptable carrier, wherein the neurodegenerativedisease is selected from the group consisting of: stroke; ischemicdamage to the nervous system; neural trauma; multiple sclerosis;HIV/AIDS dementia complex; axonomy; diabetic neuropathy; Alzheimer'sdisease; Parkinson's disease; Huntington's disease; bacterial,parasitic, fungal, or viral meningitis; encephalitis; vascular dementia;multi-infarct dementia; Lewy body dementia; frontal lobe dementia;subcortical dementias; a focal cortical atrophy syndrome; ametabolic-toxic dementia; and dementia caused by an infection.
 9. Amethod for treating inflammation in a patient, the method comprisingadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of a crystalline compound having astructure of Formula (II):

wherein the compound has 2θ values 8.94; 9.39; 9.76; 10.60; 11.09;12.74; 15.27; 17.74; 18.96; 20.58; 20.88; 21.58; 21.78; 22.25; 22.80;24.25; 24.66; 26.04; 26.44; 28.32; 28.96; 29.65; 30.22; 30.46; 30.78;32.17; 33.65; 34.49; 35.08; 35.33; 37.85; and 38.48; and apharmaceutically acceptable carrier, wherein the inflammation isselected from the group consisting of: psoriasis, dermatitis; systemicscleroderma, sclerosis; inflammatory bowel disease; respiratory distresssyndrome; meningitis; encephalitis; uveitis; colitis;glomerulonephritis; an allergic condition; atherosclerosis; leukocyteadhesion deficiency; diabetes mellitus; central nervous system (CNS)inflammatory disorder; allergic encephalomyelitis; and allergicneuritis.
 10. A method for treating a fibrotic-associated condition in apatient, the method comprising administering to the patient apharmaceutical composition comprising a therapeutically effective amountof a crystalline compound having a structure of Formula (II):

wherein the compound has 2θ values 8.94; 9.39; 9.76; 10.60; 11.09;12.74; 15.27; 17.74; 18.96; 20.58; 20.88; 21.58; 21.78; 22.25; 22.80;24.25; 24.66; 26.04; 26.44; 28.32; 28.96; 29.65; 30.22; 30.46; 30.78;32.17; 33.65; 34.49; 35.08; 35.33; 37.85; and 38.48; and apharmaceutically acceptable carrier, wherein the fibrotic-associatedcondition is selected from the group consisting of cystic fibrosis,injection fibrosis, endomyocardial fibrosis, idiopathic pulmonaryfibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massivefibrosis, nephrogenic systemic fibrosis, cirrhosis, diffuse parenchymallung disease, post-vasectomy pain syndrome, tuberculosis, sickle-cellanemia and rheumatoid arthritis.
 11. A method for treating anischemic-related condition in a patient, the method comprisingadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of a crystalline compound having astructure of Formula (II):

wherein the compound has 2θ values 8.94; 9.39; 9.76; 10.60; 11.09;12.74; 15.27; 17.74; 18.96; 20.58; 20.88; 21.58; 21.78; 22.25; 22.80;24.25; 24.66; 26.04; 26.44; 28.32; 28.96; 29.65; 30.22; 30.46; 30.78;32.17; 33.65; 34.49; 35.08; 35.33; 37.85; and 38.48; and apharmaceutically acceptable carrier, wherein the ischemic-relatedcondition is selected from the group consisting of: acute coronarysyndrome, arterial occlusive disease, atherosclerosis, infarction, heartfailure, pancreatitis, myocardial hypertrophy, stenosis, and restenosis.12. The method of claim 11, wherein the ischemic-related condition isheart failure.
 13. A method for treating an infection in a patient, themethod comprising administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount of acrystalline compound having a structure of Formula (II):

wherein the compound has 2θ values 8.94; 9.39; 9.76; 10.60; 11.09;12.74; 15.27; 17.74; 18.96; 20.58; 20.88; 21.58; 21.78; 22.25; 22.80;24.25; 24.66; 26.04; 26.44; 28.32; 28.96; 29.65; 30.22; 30.46; 30.78;32.17; 33.65; 34.49; 35.08; 35.33; 37.85; and 38.48; and apharmaceutically acceptable carrier, wherein the infection is aparasitic infection caused by a protozoan parasite selected from thegroup consisting of: Plasmodium sps., Trypanosoma sps., Leishmania sps.,Pneumocystis carinii, Toxoplasma gondii, Entamoeba histolytica,Entamoeba invadens, and Giardia lamblia.
 14. A method for treatingosteoporosis in a patient, the method comprising administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective amount of a crystalline compound having a structure of Formula(II):

wherein the compound has 2θ values 8.94; 9.39; 9.76; 10.60; 11.09;12.74; 15.27; 17.74; 18.96; 20.58; 20.88; 21.58; 21.78; 22.25; 22.80;24.25; 24.66; 26.04; 26.44; 28.32; 28.96; 29.65; 30.22; 30.46; 30.78;32.17; 33.65; 34.49; 35.08; 35.33; 37.85; and 38.48; and apharmaceutically acceptable carrier.